high fluence low-power laser irradiation induces apoptosis via inactivation of akt/gsk3β signaling...

High Fluence Low-Power LaserIrradiation Induces Apoptosisvia Inactivation of Akt/GSK3bSignaling PathwayLEI HUANG, SHENGNAN WU, AND DA XING*

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University,

Guangzhou 510631, China

High fluence low-power laser irradiation (HF-LPLI) is a newly discovered stimulus through generating reactive oxygen species (ROS) totrigger cell apoptosis. Activation of glycogen synthase kinase 3b (GSK3b) is proved to be involved in intrinsic apoptotic pathways undervarious stimuli. However, whether the proapoptotic factor GSK3b participates in HF-LPLI-induced apoptosis has not been elucidated.Therefore, in the present study, we investigated the involvement of GSK3b in apoptosis under HF-LPLI treatment (120 J/cm2, 633 nm).We found that GSK3b activation could promote HF-LPLI-induced apoptosis, which could be prevented by lithium chloride (a selectiveinhibitor of GSK3b) exposure or by GSK3b-KD (a dominant-negative GSK3b) overexpression. We also found that the activation ofGSK3b by HF-LPLI was due to the inactivation of protein kinase B (Akt), a widely reported and important upstream negative regulator ofGSK3b, indicating the existence and inactivation of Akt/GSK3b signaling pathway. Moreover, the inactivation of Akt/GSK3b pathwaydepended on the fluence of HF-LPLI treatment. Furthermore, vitamin c, a ROS scavenger, completely prevented the inactivation of Akt/GSK3b pathway, indicating ROS generation was crucial for the inactivation. In addition, GSK3b promoted Bax activation by down-regulating Mcl-1 upon HF-LPLI treatment. Taken together, we have identified a new and important proapoptotic signaling pathway that isconsisted of Akt/GSK3b inactivation for HF-LPLI stimulation. Our research will extend the knowledge into the biological mechanismsinduced by LPLI.J. Cell. Physiol. 226: 588–601, 2011. � 2010 Wiley-Liss, Inc.

High fluence low-power laser irradiation (HF-LPLI) caninterfere with cell cycle and inhibit cell proliferation, whichcould be used to control certain types of hyperplasia (Gross andJelkmann, 1990;O’Kane et al., 1994). This research is consistentwith the results obtained by Zhang et al. (2008), which revealsthatwhen light irradiation fluence exceeds 25 J/cm2, a significantsuppression of cell viability is observed inHeLa cells. Previously,we have demonstrated that HF-LPLI induces human lungadenocarcinoma (ASTC-a-1) cells apoptosis by activatingcaspase-3 with fluence�60 J/cm2 (Wang et al., 2005;Wu et al.,2007). We also demonstrate that HF-LPLI (80, 120 J/cm2)triggers cell apoptosis through mitochondrial oxidative damageindicated by the collapse of mitochondrial transmembranepotential (Dcm; Wu et al., 2007). Subsequently, we ascertainthat HF-LPLI induces cell apoptosis via cyclosporine A-sensitivemitochondrial permeability transition, and cytotoxic doses ofreactive oxygen species (ROS) generation has beendemonstrated to be a determinant of cell apoptosis (Wu et al.,2009). However, the mechanism of apoptosis caused byHF-LPLI has not been fully identified.

Glycogen synthase kinase 3b (GSK3b) is a serine/threoninekinase that regulates many fundamental cellular functionsincluding cell cycle, microtubule dynamics, protein synthesis,gene expression, and apoptosis (Grimes and Jope, 2001).GSK3b exerts its actions by directly phosphorylating varioussubstrates including cyclin D (Diehl et al., 1998), Tau (Hangeret al., 1992), translation factor eukaryotic initiation factor 2B(Pap and Cooper, 2002), c-Jun (de Groot et al., 1993), c-Myc(He et al., 1998), and b-catenin (Ciani and Salinas, 2005).Although GSK3b is considered to be a constitutively activeenzyme, its activity is tightly controlled by its phosphorylationlevels. Akt activation is often effective forGSK3b inactivation byincreasing the serine-9 phosphorylation (Cross et al., 1995). Inaddition, several other signaling pathways are also involved inthemodulation of GSK3b activity, such asWnt pathway (Frame

and Cohen, 2001; Doble and Woodgett, 2003) and MAPK/p90Rsk pathway (Stambolic and Woodgett, 1994).

Accumulated evidences suggest that GSK3b is an upstreamregulator of apoptosis and possesses proapoptoticcharacteristics. GSK3b promotes cell apoptosis caused byoxidative stress (Shin et al., 2004), DNA damage (Tan et al.,2006), endoplasmic reticulum stress (Kim et al., 2005), andceramide (Mora et al., 2002). In addition, the catalyticallyinactive mutant of GSK3b or treatment with inhibitors of

Abbreviations: ASTC-a-1, human lung adenocarcinoma cells; COS-7,African green monkey SV-40-transformed kidney fibroblast cells;GSK3b, glycogen synthase kinase 3b; HepG2, humanhepatocellular liver carcinoma cells; H2DCFDA,dichlorodihydrofluorescein diacetate; HF-LPLI, high fluence low-power laser irradiation; LPLI, low-power laser irradiation; NAC,N-acetylcysteine; ROS, reactive oxygen species; Vc, vitamin c.

Contract grant sponsor: National Basic Research Program ofChina;Contract grant number: 2010CB732602.Contract grant sponsor: Program for Changjiang Scholars andInnovative Research Team in University;Contract grant number: IRT0829.Contract grant sponsor: National Natural Science Foundation ofChina;Contract grant numbers: 30870676, 30870658.

*Correspondence to: Da Xing, MOE Key Laboratory of Laser LifeScience & Institute of Laser Life Science, College of Biophotonics,South China Normal University, Guangzhou 510631, China.E-mail: [email protected]

Received 30 April 2010; Accepted 27 July 2010

Published online in Wiley Online Library(wileyonlinelibrary.com), 3 August 2010.DOI: 10.1002/jcp.22367

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GSK3b, such as lithium chloride (LiCl), protects cells fromsome apoptotic stimuli (Pap and Cooper, 1998; Hetman et al.,2000). Moreover, the inhibition of GSK3b through thephosphorylation of the serine-9 can reduce apoptosis (Li et al.,2000). It has been proposed that inhibition of GSK3b by thePI3K/Akt signaling pathway may lead to the anti-apoptoticeffects of Akt (Pap and Cooper, 1998; Wang et al., 2002).However, whether the proapoptotic factor GSK3b is involvedin HF-LPLI-induced apoptosis has not been investigated, so it isof notable consequence to bring to light whether GSK3b isactivated following HF-LPLI and, if so, how that activation isoriginated.

In this study, using flow cytometry, single-moleculefluorescence imaging and Western blot analysis, we examinedthe proapoptotic functions of GSK3b underHF-LPLI treatmentand discussed the correlation mechanisms. We tried toestablish the view that the inactivation of Akt/GSK3b signalingpathway played an important role on HF-LPLI-inducedapoptosis. Our findings will extend the knowledge of cellularsignaling mechanisms of HF-LPLI-induced apoptosis.

Materials and MethodsCell culture

ASTC-a-1 cells, HeLa cells, human hepatocellular liver carcinoma(HepG2) cells, and African green monkey SV-40-transformedkidney fibroblast (COS-7) cells were grown on 22-mm cultureglasses, in Dulbecco’s modified Eagle’s medium (DMEM; LifeTechnologies, Inc., Grand Island, NY) containing 10% fetal bovineserum (FBS; GIBCO, Co. Ltd., Grand Island, NY), 50 units/mlpenicillin and 50mg/ml streptomycin. Cells were maintained in ahumidified, 378C incubator with 5% CO2 and 95% air.

Plasmids, reagents, and antibodies

pEYFP-GSK3b, pCGN-GSK3b-WT (wild type), and pCGN-GSK3b-KD (dominant-negative GSK3b) were kindly provided byProf. John H. Kehrl (Shi et al., 2006) and Prof. Mien-Chie Hung(Ding et al., 2007), respectively. pDsRed-mit and pCFP-Bax werekindly provided by Prof. Yukiko Gotoh (Tsuruta et al., 2002) andProf. Gilmore (Valentijn et al., 2003), respectively. Hemagglutininepitope (HA)-tagged constitutively active Akt (Myr-Akt) anddominant-negative Akt (DN-Akt) were presented by Prof. Jin Q.Cheng (Yang et al., 2007).

Staurosporine (STS; 1mM), LiCl (20mM), hydrogen peroxide(H2O2; 1mM), N-acetylcysteine (NAC; 5mM) and vitamin c (Vc;100mM) were procured from Sigma (St. Louis, MO). Wortmannin(100 nM) was procured fromBIOMOLResearch Laboratories, Inc.(Plymouth, PA).

Anti-phospho-Akt (Thr308) antibody, anti-phospho-Akt(Ser473) antibody, anti-phospho-GSK3b (Ser9) antibody, anti-Aktantibody, anti-GSK3b antibody, anti-Bax antibody, and anti-caspase-3 antibody were acquired from Cell Signaling Technology(Beverly, MA). Anti-Mcl-1 antibody and anti-b-actin antibody wereacquired from Santa Cruz Biotechnology (Santa Cruz, CA). Finally,HA tag antibody was obtained from Sigma.

Cell transfection and HF-LPLI treatment

Transient transfectionwas carried out using LipofectamineTM 2000(Invitrogen, Carlsbad, CA) reagent according to manufacturer’srecommendations. Cells were seeded on 22-mm culture glasses or60-mm plates 1 day prior to transfection. The total amount ofplasmids for transfection was 0.5 or 5mg. Cells were maintained inserum-free medium during transfection, and replaced with freshculture medium 6 h later. After 24 h expression, cells weresubjected to different treatments.

For irradiation of a single cell, the experiment was conducted asdescribed in our previous work (Wu et al., 2009). Laser irradiationwas performed through the objective lens (40�/NA1.45) of the

inverted microscope (LSM510-ConfoCor2; Zeiss, Jena, Germany)in laser scanning mode. The cells in the selected area wereirradiation for 10min with a fluence of 120 J/cm2. The powerintensity was maintained at 0.2W/cm2. For irradiation of multiplecells, the cells were irradiated with a He–Ne laser (632.8 nm, HN-1000; Guangzhou, China) for 1.66, 3.33, 6.66, 10, 13.33min in thedark with the corresponding fluence of 20, 40, 80, 120, and 160 J/cm2, respectively. The irradiation light fluence rate was againmaintained at 0.2W/cm2.

Flow cytometry

For FACS analysis, Annexin-V-FITC conjugate, PI dyes, and bindingbuffer were used as standard reagents. Flow cytometry wasperformed using a FACScanto II flow cytometer (BectonDickinson, Mountain View, CA) with excitation at 488 nm.Fluorescent emission of FITC was measured at 515–545 nm andthat of DNA–PI complexes at 564–606 nm. Cell debris wasexcluded from the analysis by an appropriate forward light scatterthreshold setting. Compensation was used wherever necessary.

ROS measurements

To quantify ROS generation caused by HF-LPLI, HF-LPLI plusVc, H2O2 or HF-LPLI plus H2O2, ASTC-a-1 cells were incubatedwith 10mM H2DCFDA (Invitrogen) for 30min at 378C. Afterincubation, cells were washed with PBS, trypsinized, andresuspended in PBS solution. DCF fluorescence (the fluorescentproduct of H2DCFDA) was measured using FACScanto II flowcytometer (excitation at 488 nm, emission at 515–545 nm) anddatawere analyzed with CELL Quest software (Becton Dickinson).

Bax gene silencing by shRNA

RNA interference of Baxwas performed using Bax-shRNA plasmid(p-Genesil-3-Bax) purchased fromGenesil Biotechnology (Wuhan,China). The following sequences were targeted to silence Bax byshRNA expression: AACATGGAGCTGCAGAGGATGAdTdT.Also, p-Genesil-3 containing non-specific shRNA was used as anegative control (p-Genesil-3-con). The targeting sequences ofnon-specific shRNA were CTGAAGTATTCCGCGTACG. Fortransfection, ASTC-a-1 cells were seeded in 60-mm plates at 50%confluency, and then the cells were transfected with 2mg p-Genesil-3-Bax and p-Genesil-3-con, respectively, usingLipofectamineTM 2000 according to Invitrogen transfectionprotocols. After transfection, transfected cells (colonies) wereselected by culture with G418 (Sigma; for ASTC-a-1 cells, 800mg/ml for 1 week). The efficiency of Bax-shRNA knockdown wasidentified by Western blot analysis.

Cell lysates collection

ForWestern blot analysis, cells in 60-mmplateswerewashed threetimes with phosphate-buffered saline (PBS) and were lysed with150ml of lysis buffer (20mM Tris, pH 7.4, 150mM NaCl, 2mMEDTA, 2mM EGTA, 1mM sodium orthovanadate, 50mM sodiumfluoride, 1% TritonX-100, 0.1% SDS and 100mMphenylmethylsulfonyl fluoride). For caspase-3 activity assay, cellswere lysed with 150ml lysis buffer without sodium orthovanadate.The lysates were collected in microcentrifuge tubes andcentrifuged. Protein concentrations were determined using theBradford method. The lysates were stored at�808C forWesternblot.

Western blot analysis

Cell lysates were mixed with Laemmli sample buffer (2% SDS) andplaced in boiling water for 10min. Proteins were separated in 15%SDS–polyacrylamide gels for Bax and caspase-3, and in 12% SDS–polyacrylamide gels for phospho-Thr308-Akt, phospho-Ser473-Akt, total Akt, HA-Akt, phospho-Ser9-GSK3b, total GSK3b,Mcl-1, andb-actin. The proteins were transferred to nitrocellulosemembranes and incubated with primary antibodies of Bax

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(1:1,000), caspase-3 (1:1,000), phospho-Thr308-Akt (1:1,000),phospho-Ser473-Akt (1:1,000), total Akt (1:1,000), HA (1:1,000),phospho-Ser9-GSK3b (1:1,000), total GSK3b (1:1,000), Mcl-1(1:500) or b-actin (1:1,000). Bax, Caspase-3, phospho-Ser473-Akt,total Akt, HA-Akt, phospho-Ser9-GSK3b, and total GSK3b werelabeled with goat anti-rabbit conjugated to IRDyeTM 800secondary antibodies (Rockland Immunochemicals, Gilbertsville,PA). Phospho-Thr308-Akt, Mcl-1, and b-actin were labeledwith goat anti-mouse conjugated to Alexa Flour 680 secondaryantibodies (Invitrogen). Detection was performed using the

LI-COR Odyssey Scanning Infrared Fluorescence Imaging System(LI-COR, Inc., Lincoln, NE).

Immunofluorescence

For Bax activation, ASTC-a-1 cells cultured on glass cover slipswere stained with Mito-Tracker Deeper 633 Red (100 nM;Molecular Probes, Inc.) at 378C for 30min. Then the cells werefixed with 4% paraformaldehyde for 10min, followed bypermeabilization in 0.5% CHAPS for 30min. The cells were

Fig. 1. GSK3bpromotes cell apoptosis in response toHF-LPLI stimulation.ASTC-a-1 cellswere transfectedwithpGSK3b-WT,pGSK3b-KD,orpre-treated with LiCl (20mM) for 24h. The cells were treated with HF-LPLI (120 J/cm2) except for control group. A: Representative sequentialimagesof apoptoticmorphologyof treated cellswereobservedwithHoechst 33258 staining.Thecellswerephotographedat 40Tmagnifications.Data representmeanWSEM (nU 3). B,C:Cell death analysis of treated cells was performedby flow cytometrywith annexin V/PI double staining.Representative images and quantitative analysis were shown in (B) and (C), respectively. Data representmeanWSEM (nU 3;

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P<0.05 vs. controlcells, #P<0.05 vs. indicated cells). D: Representative Western blot analysis of treated cells was performed to detect protein levels of full lengthcaspase-3 and cleaved caspase-3. E: Quantitative analysis of cleaved caspase-3 protein levels in treated cells. Data representmeanWSEM (nU 3;M

P<0.05 vs. control cells, #P<0.05 vs. indicated cells). [Color figure canbe viewed in the online issue,which is available atwileyonlinelibrary.com.]


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incubated in blocking buffer (10% bovine serum albumin in PBS) for1 h at room temperature, followed by incubation with anti-Baxantibody 6A7 (diluted 1:30 in blocking buffer; Abcam, Cambridge,United Kingdom) at 48C overnight. Samples were washed threetimes for 5min in PBS, and then incubated for 1 h with Alexa Fluor488-conjugated goat anti-mouse secondary antibody (diluted 1:300

in blocking buffer; Invitrogen) at room temperature. Images wereacquired using a confocal microscope through a 40� oil objective(LSM510-ConfoCor2; Zeiss).

Statistical analysis

MATLAB software was used for data analysis. For fluorescenceemission intensity analysis, a background subtraction wasperformed for the data. For the analysis of Bax translocation,images were analyzed with MATLAB 6.5 software by drawingregions around individual cells and then computing standarddeviations of the intensity of the pixels (punctate/diffuse) andintegrated brightness (total brightness; Munoz-Pinedo et al., 2006).

All assays were repeated independently for a minimum of threetimes. Data are represented asmean� SEM. Statistical analysis wasperformed with Student’s paired t-test. Differences wereconsidered statistically significant at P< 0.05.

ResultsGSK3b promotes cell apoptosis induced by HF-LPLI

Nuclear staining by Hoechst 33258 dyes was used to monitorthe morphological changes of the nucleus in cells underdifferent treatments. Nuclear condensation and cell shrinkagerevealed the characteristic morphology associated withapoptosis. ASTC-a-1 cells were divided into five groups. Twoofthe five groups with GSK3b-WT and GSK3b-KDoverexpression, respectively, were treated with HF-LPLI.Another group was incubated with GSK3b inhibitor LiCl for24 h before HF-LPLI treatment. Cells without any treatmentwere set as control. The last one of the five groups was treatedwith HF-LPLI alone. Cells were stained with Hoechst 33258dyes 4 h post HF-LPLI treatment and then monitored byconfocal microscopy. The representative fluorescence imagesof cells labeled with Hoechst 33258 under various treatmentswere shown in Figure 1A. The results revealed that all theHF-LPLI-treated cells under different conditions displayedobvious nuclear condensation and chromatin fragmentation,confirming the occurrence of cell apoptosis in comparison withcontrol cells. The details were as follows: GSK3b-WToverexpression resulted in higher levels of cell apoptosis thanHF-LPLI treatment alone. In contrast, GSK3b-KDoverexpression resulted in lower levels of cell apoptosis thanHF-LPLI treatment alone. Besides, LiCl exposure resulted insimilar levels of cell apoptosis with the condition of GSK3b-KDoverexpression. Quantitative analysis (Fig. 1B,C) of cell deathrate by flow cytometry revealed that all the HF-LPLI-treatedcells under different conditions showed cell death incomparison with control cells. Specifically, GSK3b-WToverexpression resulted in higher rates of cell death thanHF-LPLI treatment alone. In contrast, GSK3b-KDoverexpression resulted in lower rates of cell death than HF-LPLI treatment alone. Besides, LiCl exposure resulted in similarrates of cell death with the condition of GSK3b-KDoverexpression. These results demonstrate that GSK3bpromotes cell apoptosis induced by HF-LPLI.

Subsequently, activity of caspase-3 under differenttreatments was examined withWestern blot analysis (Fig. 1D).Our results showed that cleaved caspase-3 was detected in allcells treated with HF-LPLI in comparison with the control cells.For the details, GSK3b-WT overexpression resulted in higherlevels of cleaved caspase-3 than HF-LPLI treatment alone(Fig. 1E). In contrast, cells with GSK3b-KD overexpressionresulted in similar levels of cleaved caspase-3 with LiClexposure. Both GSK3b-KD overexpression and LiCl exposureresulted in lower levels of cleaved caspase-3 than HF-LPLItreatment alone. The results shown in Figure 1D,E demonstratethat GSK3b promotes cell apoptosis (indicated by theenhanced activity of caspase-3) induced by HF-LPLI.

Fig. 1. (Continued )



HF-LPLI induces GSK3b nuclear translocation

A powerful method for imaging and quantifying the spatio-temporal nuclear translocation of GSK3b in living cells is thetransfection of cells with YFP-GSK3b reporter. To assess theeffect of HF-LPLI on GSK3b nuclear translocation, YFP-GSK3breporter was transfected into ASTC-a-1 cells and COS-7 cells,and the dynamic changes of YFP-GSK3b was monitored inreal-time in living cells under different treatments by confocalmicroscope. The results showed that HF-LPLI (120 J/cm2)caused a marked 2.4-fold within 8 h increase in YFP-GSK3bnuclear fluorescence emission intensities, compared to that inthe control cells (Fig. 2A,B), revealing the nuclear translocationof GSK3b. The phenomenon of GSK3b nuclear translocation isconsistent with that caused by STS or wortmannin treatment,the conditions known to inactivateAkt, indicating the possibilityof Akt involves in GSK3b activation under HF-LPLI treatment.Similar results were observed in COS-7 cells (Fig. 2C,D).

HF-LPLI induces GSK3b activation through Aktinactivation

We next investigated whether GSK3b activation could bereliably initiated by Akt inactivation under HF-LPLI treatment.Phosphorylation levels of Akt and GSK3b in ASTC-a-1 cellsunder HF-LPLI (120 J/cm2) treatment were identified withWestern blot analysis. Date shown in Figure 3A–C revealedthat HF-LPLI caused significant reduction of the levels of

phospho-Thr308-Akt, phospho-Ser473-Akt, and phospho-Ser9-GSK3b, which were positively correlated with irradiationtime, whereas total levels of Akt and GSK3b were unchanged.STS and wortmannin were set as positive controls.Wortmannin reduced both the levels of phospho-Thr308-Aktand phospho-Ser473-Akt, while STS only reduced the levels ofphospho-Thr308-Akt. Experiments performed in both HeLacells and HepG2 cells yielded similar results that HF-LPLIreduced the levels of phospho-Thr308-Akt, phospho-Ser473-Akt, and phospho-Ser9-GSK3b (Fig. 3D–F). These resultsdemonstrate that HF-LPLI causes significant inactivation of Aktand activation of GSK3b indicated by the decrease inphosphorylation levels, suggesting that HF-LPLI inducesinactivation of the Akt/GSK3b signaling pathway.

In order to further confirm the result that HF-LPLI causedGSK3b activation through Akt inactivation, the ASTC-a-1 cellswere divided into five groups. Two of the five groups withMyr-Akt and DN-Akt overexpression, respectively, weretreated with HF-LPLI (120 J/cm2). Another group was treatedwith wortmannin alone. Cells without any treatment were setas control. The last group was treated with HF-LPLI alone.Changes in the levels of phospho-Ser9-GSK3b and theexogenous expression of Myr-Akt and DN-Akt were detectedusing Western blot analysis 4 h post HF-LPLI treatment. Theresults revealed that overexpression of Myr-Akt resulted inhigher levels of phospho-Ser9-GSK3b than HF-LPLI treatmentalone (Fig. 3G,H). In contrast, overexpression of DN-Akt

Fig. 2. HF-LPLI induces GSK3b nuclear translocation. A,C: Representative sequential images of ASTC-a-1 cells (A) and COS-7 cells (C)expressingYFP-GSK3b (yellowemission)was inresponsetodifferenttreatments;BarU 10mm.B,D:Quantitativeanalysisof relativeYFP-GSK3bfluorescence emission intensities of nucleus in ASTC-a-1 cells (B) and in COS-7 cells (D) was subjected to different treatments. Data representmeanWSEM (nU 5). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


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Fig. 3. HF-LPLIinducesactivationofGSK3bthroughinactivationofAkt.A:WesternblotanalysisofASTC-a-1cellsreceiveddifferenttreatmentswas performed to detect phospho-Thr308-Akt, phospho-Ser473-Akt, phospho-Ser9-GSK3b, total Akt, and total GSK3b. Data were therepresentativegraph.B,C:Quantitativeanalysisof the levelsofphospho-Thr308-Akt,phospho-Ser473-Akt,andphospho-Ser9-GSK3b inASTC-a-1 cells received different treatments. Data representmeanWSEM (nU 3;

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P<0.05 vs. 0 h). D:Western blot analysis ofHeLa cells andHepG2 cellstreatedwithHF-LPLI(120 J/cm2)wasperformedtodetectphospho-Thr308-Akt,phospho-Ser473-Akt,phospho-Ser9-GSK3b, totalAkt,andtotalGSK3b. Datawere the representative graph. E,F:Quantitative analysis of the levels of phospho-Thr308-Akt, phospho-Ser473-Akt, and phospho-Ser9-GSK3b inHeLacellsandHepG2cellstreatedwithHF-LPLI(120 J/cm2).DatarepresentmeanWSEM(nU 3;

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P<0.05vs.0 h).G:ASTC-a-1cellsexpressing Myr-Akt or DN-Akt were treated with HF-LPLI (120 J/cm2). Cells without any treatment were set as control. Wortmannin wasused as a positive control. The graph was representative Western blot analysis of phospho-Ser9-GSK3b in cells received different treatments.H: Quantitative analysis of the levels of phospho-Ser9-GSK3b in ASTC-a-1 cells received different treatments. Data represent meanWSEM(nU 3;

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P<0.05 vs. indicated cells). I: Representative fluorescence images ofASTC-a-1 cells expressingYFP-GSK3b (yellowemission) in responseto different treatments; BarU 50mm. J: The percentages of ASTC-a-1 cells with YFP-GSK3b nuclear translocation in response to differenttreatments.DatarepresentmeanWSEMoffive independentexperimentswith500cellsperconditions (

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P<0.05vs. indicatedcells).K:Exogenousexpression of Myr-Akt and DN-Akt in ASTC-a-1 cells were detected usingWestern blot analysis. [Color figure can be viewed in the online issue,which is available at wileyonlinelibrary.com.]



resulted in lower levels of phospho-Ser9-GSK3b than HF-LPLItreatment alone. Wortmannin was set as a positive control todecrease the levels of phospho-Thr308-Akt and phospho-Ser473-Akt. These results confirm that HF-LPLI causes GSK3bactivation through Akt inactivation.

We also explored whether Akt had effect onGSK3b nucleartranslocation induced byHF-LPLI. To this end, YFP-GSK3bwasco-expressed with Myr-Akt or DN-Akt in ASTC-a-1 cells andthen the cells were treated with HF-LPLI (120 J/cm2). Cellsexpressing YFP-GSK3b alone without any treatment were setas control. The representative fluorescence images of YFP-GSK3b nuclear translocation in cells under HF-LPLI treatmentwere shown in Figure 3I, and the quantitative analysis of YFP-GSK3b nuclear translocation was shown inFigure 3J. Exogenous expression of Myr-Akt and DN-Akt wereidentified using Western blot analysis (Fig. 3K). We found thatMyr-Akt overexpression resulted in lower percentages of YFP-GSK3b nuclear translocation than HF-LPLI treatment alone. Incontrast, DN-Akt overexpression resulted in higherpercentages of YFP-GSK3b nuclear translocation than HF-LPLItreatment alone. There was little YFP-GSK3b nucleartranslocation in control cells. These results suggest that Akt

regulates GSK3b nuclear translocation in response to HF-LPLIstimulation.

HF-LPLI induces inactivation of the Akt/GSK3b signalingpathway in a dose-dependent manner

Next, changes in the activities of Akt and GSK3b underdifferent fluences of HF-LPLI treatment were investigated inASTC-a-1 cells. The phosphorylation levels of Akt and GSK3bwere examined with Western blot analysis 4 h post HF-LPLItreatment at the fluence of 20–160 J/cm2.Concomitantwith theincrease of HF-LPLI fluence, the levels of phospho-Thr308-Akt,phospho-Ser473-Akt, and phospho-Ser9-GSK3b weredecreased whereas the total levels of Akt and GSK3b wereunchanged (Fig. 4A–C). These results demonstrate thatHF-LPLI induces inactivation of the Akt/GSK3b signalingpathway in a dose-dependent manner.

ROS mediates inactivation of the Akt/GSK3b signalingpathway under HF-LPLI treatment

To examine whether ROS contributed to the activation ofGSK3b during HF-LPLI-induced apoptosis, ASTC-a-1 cells



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were transfected with YFP-GSK3b and the dynamic changes ofYFP-GSK3b were monitored under various treatments. Therepresentative fluorescence images of YFP-GSK3b in cellsunder different treatments were shown in Figure 5A, and thequantitative analysis of YFP-GSK3b fluorescence emissionintensities of nucleus was shown in Figure 5B. Intracellular ROSgeneration was quantified by DCF fluorescence under varioustreatments (Fig. 4D). Our experiments revealed that nucleartranslocation of YFP-GSK3b under HF-LPLI (120 J/cm2)treatment was slower than that under combined treatments ofHF-LPLI and H2O2 (Fig. 5A,B). In marked contrast, Vc or NAC,ROS scavenger, significantly decreased HF-LPLI-inducedtranslocation of YFP-GSK3b. In addition, the percentages ofcells with YFP-GSK3b nuclear translocation 4 h after eachtreatment were calculated as follows (n¼ 5, 500 cells percondition): HF-LPLI caused 81% translocation; H2O2 caused86% translocation; HF-LPLI combined with H2O2 caused 98%translocation; HF-LPLI combined with Vc or NAC caused 15%and 29% translocation, respectively, suggesting ROS generationwas involved in the activation of GSK3b.

In order to explore whether ROS mediated activation ofGSK3b through inactivation of Akt under HF-LPLI treatment,the phosphorylation levels of Akt and GSK3b under differenttreatments were detected with Western blot analysis. Asshown in Figure 5E–G, the phosphorylation levels of Akt andGSK3b under HF-LPLI (120 J/cm2) treatment were higher thanthat under combined treatments of HF-LPLI and H2O2. Incontrast, the levels under HF-LPLI treatment were lowerthan those under HF-LPLI treatment in the presence of Vc.The total levels of Akt and GSK3b were unchanged. Theseresults demonstrate that HF-LPLI induces inactivation of theAkt/GSK3b pathway mediated by ROS generation.

GSK3b promotes Bax activation through down-regulation of Mcl-1 under HF-LPLI treatment

To understand the effects of GSK3b on Bax activation underHF-LPLI treatment, ASTC-a-1 cells co-expressing CFP-Bax,YFP-GSK3b, and DsRed-mit were treated with HF-LPLI(120 J/cm2). Fluorescence images and quantitative analysis offluorescence emission intensities of CFP-Bax and YFP-GSK3brevealed that nuclear translocation ofGSK3boccurred prior tomitochondrial translocation of Bax under HF-LPLI treatmentin comparison with control cells (Fig. 6A–D), suggesting thatGSK3b could promote Bax activation.

To confirm the result that GSK3b promoted Bax activationunder HF-LPLI treatment, immunofluorescence technologywas used to detect Bax activation with Bax 6A7 antibody withor without LiCl exposure or overexpression of GSK3b-KD.Figure 6E showed that HF-LPLI induced high levels of activatedBax compared with the control cells 8 h post-treatment.Conversely, LiCl pre-treatment or overexpression of GSK3b-KD significantly suppressed Bax activation. These resultsindicate that GSK3b activation is an upstream event ofBax activation under HF-LPLI treatment.

We also explored whether HF-LPLI-induced cell apoptosiswas neutralized by Bax knockdown. ASTC-a-1 cells weretransfected with Bax-shRNA. G418-resistant cells werecollected, and loss of Bax was identified by Western blotanalysis (Fig. 6F). As seen in Figure 6G, cell apoptosis analyzedusing flow cytometry revealed that HF-LPLI resulted in obviousapoptosis compared with control cells. Knockdown ofBax by shRNA significantly suppressed HF-LPLI-induced cellapoptosis, indicating that Bax activation play an important rolein the apoptotic process.

We next examined whether Mcl-1, an anti-apoptoticmember of Bcl-2 family and a known substrate of GSK3b, wasinvolved in the regulation of Bax activation under HF-LPLI

Fig. 4. HF-LPLI induces inactivation of Akt/GSK3b signalingpathway inadose-dependentmanner.A:ASTC-a-1 cellswere treatedwith HF-LPLI at the fluence of 20, 40, 80, 120, or 160 J/cm2. After 4 h,the levels of phospho-Thr308-Akt, phospho-Ser473-Akt, phospho-Ser9-GSK3b, total Akt, and total GSK3b were detected withWestern blot analysis. Data were the representative graph.B,C: Quantitative analysis of the levels of phospho-Thr308-Akt,phospho-Ser473-Akt, and phospho-Ser9-GSK3b in ASTC-a-1 cellsreceived HF-LPLI treatment at different fluences. Data representmeanWSEM (nU 3;

M

P<0.05 vs. control cells).



Fig. 5. ROSmediates inactivationofAkt/GSK3b signalingpathwayunderHF-LPLI treatment.A:Representative sequential imagesofASTC-a-1cells expressing YFP-GSK3b (yellow emission) received different treatments. BarU 10mm. B: Quantitative analysis of relative YFP-GSK3bfluorescence emission intensities of nucleus in ASTC-a-1 cells received different treatments. Data represent meanWSEM (nU 5). C: Thepercentages of ASTC-a-1 cells with YFP-GSK3b nuclear translocation received different treatments. Data represent meanWSEM of fiveindependent experimentswith 500 cells per conditions (

M

P<0.05 vs. indicated cells, #P<0.05 vs. indicated cells). GSK3bnuclear translocationwasregulated by ROS generation under HF-LPLI treatment. D: ASTC-a-1 cells were incubated with 10mM H2DCFDA for 30min at 37-C and thentreatedwithHF-LPLI, HF-LPLI plus Vc, H2O2 orHF-LPLI plusH2O2. ROS generation was determined byDCF fluorescence andmeasured usingFACS analysis. E–G: RepresentativeWestern blot analysis (E) and quantitative analysis (F,G) for the levels of phospho-Thr308-Akt, phospho-Ser473-Akt, andphospho-Ser9-GSK3b inASTC-a-1 cells receiveddifferent treatments.Data representmeanWSEM(nU 3;

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P<0.05vs. indicatedcells; Vc, Vitamin c; NAC, N-acetylcysteine). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


596 H U A N G E T A L .

Fig. 6. GSK3b promotes Bax activation through down-regulation ofMcl-1 underHF-LPLI treatment.A,C: Representative sequential images ofASTC-a-1 cells co-expressingCFP-Bax (cyanemission), YFP-GSK3b (yellowemission), andDsRed-mit (red emission)without any treatment (A)or treated with HF-LPLI (120 J/cm2) (C). BarU 10mm. B,D: Quantitative analysis of relative nuclear YFP-GSK3b and mitochondrial CFP-Baxfluorescence emission intensities in ASTC-a-1 cells without any treatment (B) or treated with HF-LPLI (120 J/cm2) (D). Data shown in B and Drepresent meanWSEM (nU 5). E: Immunofluorescence analysis of Bax activation under HF-LPLI treatment (120 J/cm2) with or without LiCl(20mM24h)exposureoroverexpressionofGSK3b-KD.Cellswithoutanytreatmentweresetascontrol.CellswerefixedandimmunostainedwithBax 6A7 antibody to determine the localization of activated Bax (green emission).Mitochondriawere labeledwithMitoTrackerDeeper Red 633(red emission). BarU 50mm (nU 3). F: ASTC-a-1 cells were transfected with either specific Bax shRNA (shRNA-Bax) or non-targeting shRNA(shRNA-nt) usingLipofectamineTM2000.G418-resistant cellswere collected, andBaxprotein expressionwas identifiedbyWesternblot analysis.G: The ASTC-a-1 cells stably transfected with shRNA-Bax were treated with HF-LPLI (120 J/cm2), and then apoptosis of treated cells wasperformed by flow cytometry with annexin V staining 8h post-treatment (nU 3). H,I: RepresentativeWestern blot analysis (H) and quantitativeanalysis (I) for the levels of phospho-Ser9-GSK3b andMcl-1 in ASTC-a-1 cells under HF-LPLI treatment (120 J/cm2) with or without LiCl (20mM24h) exposure. Data shown in I represent meanWSEM (nU 3;

M

P<0.05 vs. indicated cells). J,K: Representative Western Blot analysis (J) andquantitative analysis (K) for the levels of Mcl-1 in ASTC-a-1 cells under HF-LPLI treatment (120 J/cm2) with overexpression of GSK3b-WT andGSK3b-KD,respectively.DatashowninKrepresentmeanWSEM(nU 3;

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P<0.05vs. indicatedcells). [Colorfigurecanbeviewedintheonline issue,which is available at wileyonlinelibrary.com.]



treatment. ASTC-a-1 cells were treated with HF-LPLI with orwithout LiCl exposure. Western blot analysis revealed thatdown-regulation of Mcl-1 protein levels was concomitant withdecrease of phospho-Ser9-GSK3b levels in response to HF-LPLI (Fig. 6H and I), which were both prevented by LiCl.To further investigate the contribution made by GSK3b todown-regulation of Mcl-1, GSK3b-WT, and GSK3b-KD weretransfected into ASTC-a-1 cells, and the protein levels of Mcl-1were evaluated using Western blot analysis. As seen inFigure 6J,K, under HF-LPLI treatment, Mcl-1 protein levelswere lower in cells overexpressing GSK3b-WT than that innon-overexpressing cells. In contrast, the levels were higherin cells overexpressing GSK3b-KD than that in non-overexpressing cells, suggesting that GSK3b down-regulatedthe protein levels of Mcl-1 in response to HF-LPLI. Takentogether, results shown in Figure 6 demonstrate that GSK3bpromotes Bax activation through down-regulation of Mcl-1during HF-LPLI-induced apoptosis.

Discussion

In light of our recent report that HF-LPLI induces intrinsicpathway through mitochondrial oxidative damage, cytochrome

c release, and caspase-3 activation, in the present study, weidentified a novel signaling pathway: HF-LPLI resulted ininactivation of the Akt/GSK3b signaling pathway through ROSgeneration.We also demonstrated that inactivation of the Akt/GSK3b pathway could promote HF-LPLI-induced apoptosis byaccelerating Bax activation.

GSK3b is tightly regulated by intracellular signaling systemswhen cells undergo apoptosis in response to various stimuli.Our results revealed obvious involvement of GSK3b in cellapoptosis induced by HF-LPLI. Five demonstrative evidencescould support the view: (1) GSK3b promoted cell apoptosisunder HF-LPLI treatment (Fig. 1); (2) HF-LPLI induced GSK3bactivation and nuclear translocation through Akt inactivation(Figs. 2 and 3); (3) Inactivation of Akt/GSK3b signaling pathwaytriggered by HF-LPLI was dependent on the fluence of HF-LPLItreatment (Fig. 4); (4) ROS generation was crucial forinactivation of the Akt/GSK3b pathway (Fig. 5); (5) GSK3bpromoted Bax activation by down-regulating Mcl-1 in responseto HF-LPLI treatment (Fig. 6).

It is reported that overexpression ofmodest levels ofGSK3bin SH-SY5Y neuroblastoma cells facilitates STS- and heat shock-induced apoptosis, and this facilitation was attenuated bytreatment with LiCl (Bijur et al., 2000). Using three different



598 H U A N G E T A L .

methods, we investigated the proapoptotic functions of GSK3bupon HF-LPLI treatment. Firstly, nuclear condensation andchromatin fragmentation were clearly revealed by Hoechst33258 staining in GSK3b overexpressed cells. In addition, flowcytometry, a powerful technique for quantitative analysis,supported the view that GSK3b promoted HF-LPLI-inducedapoptosis. Finally, Western blot analysis demonstrated thatGSK3b promoted apoptosis by accelerating caspase-3activation, suggesting that GSK3b activation was an upstreamevent of HF-LPLI-induced apoptosis. Besides, inhibition ofGSK3b activation by LiCl exposure or GSK3b-KDoverexpression suppressed HF-LPLI-induced apoptosis, alsodemonstrated the proapoptotic function of GSK3b uponHF-LPLI treatment.

Apoptotic stimuli including STS and growth factorwithdrawal can cause activation and nuclear accumulationof GSK3b (Bijur and Jope, 2001), which is regulated byintracellular signaling cascades. Nuclear accumulation facilitatesinteraction of GSK3b with the substrates in the nucleus.Using live-cell in situ fluorescent imaging and Western blotanalysis, nuclear translocation of GSK3b, and decrease ofphosphorylation levels of GSK3b were observed, adequatelyconfirming the activation of GSK3b under HF-LPLI treatment.Akt is an important upstreamnegative regulator ofGSK3b.Ourexperiments corroborated that Akt was involved in theactivation of GSK3b, which was evidenced by simultaneousdecrease in the levels of phospho-Thr308-Akt, phospho-Ser473-Akt, and phospho-Ser9-GSK3b. In addition, DN-AktandMyr-Akt overexpression could promote and inhibit GSK3bactivity, respectively. Thus, we concluded that inactivation ofthe Akt/GSK3b signaling pathway was existed in HF-LPLI-induced apoptosis.

ROS play critical roles in the regulation of apoptosis (Simonet al., 2000; Chen et al., 2003). Previously, we have reportedthat the generation of cytotoxic ROS is crucial for HF-LPLI-induced apoptosis (Wu et al., 2009). However, whether ROSparticipate in the inactivation of Akt/GSK3b signaling pathwayupon HF-LPLI treatment is unclear. In the present study, Vc, aROS scavenger, completely suppressed the inactivation of Akt/GSK3b pathway under HF-LPLI treatment (Fig. 5), indicatingthe involvement of ROS in the pathway. Early reports revealthat the formation of ceramide is a key event in oxidativestress-induced apoptosis (Goldkorn et al., 1998; Bezombeset al., 2001). Moreover, ceramide can activate PP2A(Dobrowsky et al., 1993; Chalfant et al., 1999; Ruvolo et al.,1999), a protein phosphatase, which can inactivateAkt (Yamadaet al., 2001; Li et al., 2003) and thus activateGSK3b (Ivaska et al.,2002; Lin et al., 2007). In addition, our early works show thatHF-LPLI activates caspase-3 (Wang et al., 2005;Wuet al., 2007).Previous study reports that Akt is a substrate of caspase-3 andcan be cleaved by caspase-3 (Martin et al., 2002). However, inour experiments, we did not detect Akt cleavage product inresponse to HF-LPLI stimulation (Figs. 3–5), suggestingHF-LPLI-induced Akt/GSK3b singling pathway inactivation isin a caspase-3-independent manner. Therefore, we prefer thatthe HF-LPLI/ROS/ceramide/PP2A signaling pathway mediatesinactivation of the Akt/GSK3b signaling pathway.

The activity state of Akt/GSK3b signaling pathway can bemodulated by the changes of intracellular oxidant levels upondifferent stimuli. Karu (1989) have reported that LPLI canthrough activating endogenous photoacceptors and generatingROS to modulate various biological processes. Our earlierstudy shows that the level of ROS generation is positivecorrelation with the fluence of LPLI (Zhang et al., 2008). Zhanget al. (2009) have reported that LPLI at low fluence of 0.2–1.2 J/cm2 promotes cell proliferation through activation of PI3K/Aktsignaling pathway. They also demonstrate that the activationof Akt/GSK3b pathway results in obvious attenuation of STS-induced cell apoptosis (Zhang et al., 2010). In contrast, in the

present study, we showed that LPLI at high fluence (120 J/cm2)resulted in inactivation of the Akt/GSK3b pathway and thusaccelerating cell apoptosis. It was reported that lethal levels ofROS as a proapoptotic stimulus could activate PP2A(Goldbaum and Richter-Landsberg, 2002; Cicchillitti et al.,2003), whereas the sublethal levels of ROS as a physiologicalregulator could suppress PP2A (Whisler et al., 1995; Rao andClayton, 2002). Besides, PP2A could dephosphorylate Akt andcause Akt inactivation (Sato et al., 2000; Beaulieu et al., 2005;Gao et al., 2005). In addition, a recent study demonstrated thatH2O2 inhibited the phosphorylation of PDK1 and Akt in aconcentration- and time-dependent manner in both PC12 cellsand neuronal cells (Chen et al., 2010). These studies wellsupported the view that the level of ROS generation was veryimportant for the activation of Akt/GSK3b signaling pathway.Another factor for the changes of the state of Akt/GSK3bsignaling pathway lied in the different cell types. For example,Ushio-Fukai et al. (1999) has reported that H2O2 (200mM)mediates Akt activation and GSK3b inactivation in vascularsmooth muscle cells. In contrast, Nair and Olanow (2008)has reported that H2O2 (200mM) causes Akt inactivationand GSK3b activation in PC12 cells. Shin et al. (2006) hasdemonstrated that H2O2 (500mM) inhibits Akt and activatesGSK3b in NIH3T3 cells.

GSK3b is most widely reported to be involved in themitochondria-mediated intrinsic apoptotic pathway. Ourresults showed that GSK3b could promote Bax activation(Fig. 6), indicating the participation of GSK3b in the intrinsicpathway under HF-LPLI treatment. Linseman et al. (2004)have reported that GSK3b directly phosphorylates Bax andpromotes its mitochondrial localization during neuronalapoptosis. On the other hand, Hongisto et al. (2008) reportsthat the locus of GSK3b action is downstream of bim geneexpression where GSK3b activity governs Bim protein levelsrather than direct phosphorylation Bax. Recent study showsthat GSK3b can phosphorylate Mcl-1 and then leads to Mcl-1degradation mediated by E3 ligase b-TrCP (Ding et al., 2007).Our results confirmed that GSK3b promoted Bax activation

Fig. 7. A model of Akt/GSK3b signaling pathway inactivationinduced by HF-LPLI.



through down-regulation of Mcl-1 during HF-LPLI-inducedapoptosis because LiCl exposure or GSK3b-KDoverexpression significantly suppressed down-regulation ofMcl-1 and Bax activation under these conditions (Fig. 6).

In conclusion, for the first time, we demonstrated thatHF-LPLI could cause Akt/GSK3b signaling pathway inactivationthrough ROS generation (Fig. 7). The inactivation of theAkt/GSK3b pathway was crucial for cell apoptosis induced byHF-LPLI. We also demonstrated that GSK3b promoted Baxactivation through down-regulation of Mcl-1, which provided adirect linkage between GSK3b and intrinsic apoptotic cascadesduring HF-LPLI-induced apoptosis. Our research provided anew proapoptotic signaling pathway under HF-LPLI treatment.

Acknowledgments

We thank Prof. John H. Kehrl (Laboratory ofImmunoregulation, National Institute of Allergy and InfectiousDiseases, National Institutes of Health) and Prof. Mien-ChieHung (Department of Molecular and Cellular Oncology, TheUniversity of Texas M.D. Anderson Cancer Center) for kindlyproviding pEYFP-GSK3b, pCGN-GSK3b-WT, and pCGN-GSK3b-KD, respectively; Prof. Yukiko Gotoh (the Institute ofMolecular and Cellular Bioscience, University of Tokyo), andProf. Gilmore (The School of Biological Sciences, University ofManchester) for kindly providing pDsRed-mit and pCFP-Bax,respectively. We also thank Prof. Jin Q. Cheng (Department ofPathology and Cell Biology and Molecular Oncology Program,H. Lee Moffitt Cancer Center and College of Medicine,University of South Florida) for gifting HA-Myr-Akt and HA-DN-Akt. This work was supported by National Basic ResearchProgram of China (2010CB732602), the Program forChangjiang Scholars and Innovative Research Team inUniversity (IRT0829), andNationalNatural Science Foundationof China (30870676; 30870658).

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